Shahrezagamasaei, Somayeh; (2021) Contribution of stromal cells to the formation and stabilisation of blood microvessels. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Vasculogenesis, the creation of new blood vessels, occurs due to an inherent ability of endothelial cells (ECs) to self-assemble and form tubules. Additionally, many studies have shown that stromal cells, connective tissue cells of any organ, including fibroblasts (FBs), play a vital role in the formation and stabilisation of new vessels, mostly chemically by producing and secreting growth factors. However, still we lack ground understanding of the mechanical contribution of FBs to vasculogenesis. In this study, employing microfluidic platforms, we aimed to address the mechanical role of FBs in vascularisation. Applying a 7-channel microfluidic platform, we encapsulated ECs with 3 different conditions: mono-cultured (MC, embedding only ECs within a hydrogel), paracrine co-cultured (PCC, embedding ECs and FBs separately in two hydrogels) and juxtacrine co-cultured (JCC, embedding ECs mixed with FBs in a hydrogel) to investigate the impact of FB presence on formation of functional microvessels. Imaging techniques and dextran perfusion revealed that the cluster-like structures formed in the device seeded with MC and PCC conditions were not functional in terms of perfusability and permeability and deteriorated within a week while the microvessels developed in the device seeded with JCC condition were functional, well-interconnected and survived much longer (~3 weeks), indicating that the direct physical interaction between FBs and ECs is crucial for the formation of functional blood microvessels. Also, chemical perturbation of mechanotransduction genes, YAP, Src, Wnt/β-catenin, RhoA, and FAK, in both ECs and FBs, resulted in either the inhibition of microvessel formation or the development of microvessels which were significantly different in morphology, perfusability, vessel length, diameter, and coverage area compared to control microvessels, demonstrating that mechanotransduction pathways play key roles in vascularisation. Additionally, using siRNA approach, we inhibited the same genes only in FBs to examine the mechanical contribution of FBs to vascularisation. This revealed that ECs co-cultured with siRNA-inhibited FBs (excluding RhoA) retained their ability to form microvessels. However, further characterisations demonstrated that the microvessels did not resemble control microvessels in terms of permeability, perfusability, tissue stiffness, barrier function, morphology, and vessel topology. Together, these results highlight the mechanical contribution of FBs to the formation, morphogenesis and function of microvessels and suggests that FBs are intrinsic mechanical promoters and stabilisers of microvessels. Such knowledge on the mechanisms underlying the vascularisation, will be useful in further developing vascularisation strategies for organ-specific, disease-specific, and cancer-specific tissue engineering and regenerative medicine applications.

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